The present invention relates to the field of detection assays and more particularly to an improved method for detecting Cryptosporidium parvum oocysts.
Parasitic infections of the gastrointestinal tract are prevalent around the world. Many gastrointestinal parasites are transmitted by the consumption of contaminated food or water. Although gastrointestinal parasitic infections in the general population cause abdominal disorders for only a short period of time, in the immunocompromised individual, a parasitic infection can be deadly.
Cryptosporidium parvum (C. parvum) is a food or waterborne parasite that infects humans and animals causing severe intestinal distress. Since the 1970""s, C. parvum has been receiving increased world wide attention. In the early 1980""s following two outbreaks of C. parvum infections in the United Kingdom resulting in a total of 516 cases, the British government was compelled to devise a method for detecting C. parvum in water. (K. M. Shepherd et al., APPLD. AND ENVIRON. MICRO., Vol. 62, No. 4 pp. 1317-1322 (1996)). In the United States, waterborne outbreaks of C. parvum are being reported with increasing frequency. One of the latest outbreaks took place in Milwaukee, Wis. in April 1993 involving the infection of an estimated 400,000 people. (C. Drozd et al. APPLD. AND ENVIRON. MICRO., Vol. 62, No. 4 pp. 1227-1232 (1996)). Infection caused by C. parvum is particularly dangerous because it can cause prolonged diarrheal illness that may be potentially fatal for immunocompromised individuals.
C. parvum is a parasite that infects its host by invading the intestinal and urogenital systems. C. parvum organisms may be transmitted in a variety of ways including via contaminated food or water, animal to animal contact, via farm animals such as sheep and calves, or alternatively by oocysts in feces. Human infections generally result from zoonotic spread, person-to-person contact, fecal-oral contact, oral-anal contact or waterborne transmission. Although, cryptosporidiosis occurs worldwide, children, travelers to foreign countries, male homosexuals, and medical personnel caring for patients with the disease, are at particular risk. In developed countries, 1 to 4% of children with gastroenteritis harbor C. parvum oocysts; and in developing countries, 4 to 11% of such children have cryptosporidiosis. Apart from humans, Cryptosporidium infections are widespread in several other vertebrates such as mammals, reptiles and fish: and accordingly, the frequency of cryptosporidiosis is reported to be relatively high for animal handlers and veterinarian personnel.
Unlike other coccidia, C. parvum is found on the brush border of intestinal epithelium and not within deep intracellular regions. Typically, C. parvum organisms are small (2 to 6 xcexcm) spherules that inhabit the microvillus border of the intestinal epithelium arranged in rows along the brush border of the jejunum. After introduction into the intestine, C. parvum sporozoites attach to the microvilli surfaces and reproduce by schizogony (asexually). The resulting infective oocysts are passed into the intestinal lumen and passed in the feces. Following ingestion of the oocysts by another vertebrate, the oocysts release sporozoites that attach themselves to the epithelial surface and initiate a new cycle of infection.
As C. parvum organisms invade the surface of intestinal cells, the host experiences symptoms such as reduced appetite, severe diarrhea and chronic fluid loss. In normal hosts, the onset of the disease is explosive, with profuse, watery diarrhea and abdominal cramping that lasts from 4 to 14 days following exposure. The symptoms generally persist for 5 to 11 days, and then rapidly abate as remission of the parasite occurs in about 10-15 days. However, in immunocompromised individuals, (i.e. marasmic and malnourished children, individuals with congenital hypogammaglobulinemia, those receiving immunosuppressants for cancer therapy or organ transplantation, and patients with AIDS), onset of the disease is more gradual and diarrhea is more severe, with daily fluid losses of up to 15 to 20 liters. Unless the underlying immunologic defect is corrected, the diarrhea may continue persistently or remittently for life. (Merck Manual, Chapter 15 p. 237 16th ed. (1992)).
There is no effective, specific anti-C. parvum therapy available at present. Although some patients have responded positively to therapy with conventional antibiotics such as spiramycin and paromomycin, the result of infection is frequently fatal for immunocompromised individuals. In fact, cryptosporidiosis is one of the predominant causes of death in immunocompromised patients.
In light of the potential disastrous consequences of C. parvum infection, sensitive, efficient methods for detecting C. parvum contamination are necessary. In humans, the typical source of cryptosporidiosis is contaminated water, therefore safeguarding water supplies is a primary goal. The United States Environmental Protection Agency has recognized the necessity for improved detection methods by initiating the establishment of mandatory guidelines for C. parvum levels in drinking water.
Currently available detection systems indicate that C. parvum organisms are observed in xe2x80x9cspikesxe2x80x9d; meaning that levels of C. parvum in samples collected upstream and downstream, from the same source of the contamination, may not be identical when simultaneous readings are made. Consequently, C. parvum levels recorded from one location may differ significantly from readings taken from the same location minutes later. Detection of C. parvum in water is further complicated because the initial source of infection is difficult to identify. An abnormally high C. parvum concentration may be caused by water run-off from contaminated farm or pasture land, or an infant""s soiled diaper carelessly discarded into a stream.
Ideally, continuous filtration systems having the capability to capture and retain C. parvum organisms for subsequent analysis would be installed in all water supply reservoirs to allow for continuous monitoring. Unfortunately, filtration systems currently in use often have filtration cartridges that either fail to retain organisms, frequently become clogged with mud or sediment, or must be replaced or cleaned with a frequency that renders the cartridges impractical.
C. parvum detection assays presently in use are cumbersome and frequently inaccurate. For example, most assay test samples begin as crude mixtures of C. parvum oocysts separated out from mud deposits collected by filters. The oocysts are isolated by processes involving centrifugation and ultrafiltration. Separating oocysts in this manner is often tedious and inefficient since each time the test sample is spun and filtered, oocysts are lost in the process, inevitably resulting in lack of sensitivity and related inaccuracies. Another significant disadvantage of such assays is the large amount of time required for processing test samples. For example, in order to improve the optical properties of test samples for detection, oocysts must be stained. Typically, staining and subsequent detection procedures can take up to four days. Furthermore, samples can be tested only in small increments (i.e. 50 xcexcl), and the sensitivity of most currently available assays is very low. Generally at least 50,000 C. parvum oocysts per milliliter must be present for a positive detection result. Therefore, C. parvum assays currently in use are generally inefficient, inaccurate and inconsistent.
Another barrier to effective Cryptosporidium screening concerns sample turbidity. The term xe2x80x9cturbidityxe2x80x9d refers specifically to the clarity or transparency of water and the effect that any suspended particles in the water may have on this clarity. Turbidity is determined by quantifying the amount of light allowed to pass through a sample and is measured in NTUs (nephelometric turbidity units). Many source water sites of public water reservoirs, e.g. rivers and lakes, often have turbidities up to 100 NTU, whereas finished water, e.g. reservoirs for public consumption, tend to have turbidities in the range of 0 to 5 NTU.
Because it is commonly suspected that Cryptosporidium contamination occurs at source water sites, efforts have been focused on assaying samples at reservoir intakes. Several gallons of source water are pumped through filters that are rated to capture particles the size of oocysts or larger. Pumping source water in this way causes large amounts of sediment to obstruct the flow of water through filters and therefore limit the volume of water passing through the filters. The filter retentates are then eluted and assayed for the presence of microorganisms. These retentates can have turbidities up to 300,000 NTU and yield highly variable C. parvum oocyst counts by immunofluorescence assay due to the loss of oocysts that occurs in multi-step sample processing.
Oocysts present in filter eluate often tend to be washed away during processing and therefore go undetected in the final step of detection assays. Consequently, currently available methods such as immunofluorescence assays (IFA) and enzyme immunoassays (EIA), are mainly useful for detecting oocysts in xe2x80x9ccleanxe2x80x9d samples, i.e. samples that have low turbidity. Such assays are more likely to give reproducible results with clean samples than those that are considered xe2x80x9cdirtyxe2x80x9d, i.e. samples that have high turbidity.
Clinical diagnosis of cryptosporidiosis is made by recovering acid-fast oocysts from stool samples. Excretion of acid-fast oocysts is most intense during the first four days of illness but persists for the duration of diarrhea. Other assays currently in use for diagnostic purposes involve the use of formalin-ethyl acetate sedimentation or Sheather""s sugar flotation stool concentration procedure to enhance the yield of oocysts in specimens containing few oocysts. Commercial fluorescein-labeled monoclonal antibody kits also provide detection of oocysts in clinical specimens. (Merck Manual, Chapter 15 p. 237 16th ed. (1992)). The disadvantage of such clinical tests is that depending on the stage of C. parvum infection, the assays may or may not be adequately sensitive for detecting oocysts. In addition, such clinical tests generally involve a multitude of steps thereby introducing a greater likelihood of inaccuracies. Furthermore, no xe2x80x9cstandardxe2x80x9d for testing stool specimens for C. parvum has been established, and so the absolute sensitivity of currently used methods has not been assessed. (Christine L. Roberts et al., JOURN. OF CLIN. MICRO., Vol. 34 No. 9, pp. 2292-2293 (1996)). Other problems associated with C. parvum testing include extensive processing time and low test positivity rates.
In summary, existing assays for parasites such as C. parvum are irreproducible, insensitive, labor-intensive, susceptible to interference by sample turbidity, and time consuming. In addition, existing assays are not quantitative, and scientific data correlating parasite levels in drinking water with incidence and severity of disease in healthy and immunocompromised persons, are unavailable. Useful assays that enable correlation of disease-parasite levels are required for the development of environmental guidelines for safeguarding water sources against C. parvum and other parasitic infestation. What is needed therefore, is a sensitive, quantitative and reproducible assay for food or waterborne parasites that can efficiently process numerous samples without requiring a multitude of steps and subjective determinations.
An efficient and sensitive method for the detection of food or waterborne parasites, such as C. parvum oocysts, is provided. In accordance with the method, molecular markers of the parasite are solubilized, thereby allowing recognition and detection of the parasite in turbid samples. Unlike prior art assays, the results of the assay described herein are both reproducible and quantitative. In addition the assay is especially useful for detecting C. parvum oocysts present in source water such as recreational water and natural bodies of water, finished water such as community water reservoirs, biological fluid samples, or fecal samples, without interference from sample turbidity. The assay is highly sensitive, allowing for the detection of less than 50,000 oocysts per milliliter, and permits rapid sample testing.
The assay described herein includes the steps of solubilizing molecular markers, or antigens, of the parasite and detecting the antigens by immunological means. Preferably the assay is useful for detecting C. parvum in test samples by solubilizing oocyst antigens and detecting the solubilized antigen, preferably by magnetic matrix capture and electrochemiluminescence. The assay is particularly suited for parasite detection in environmental water, biological fluid samples, and fecal samples because it can operate in both low and high turbidity samples, has high reproducibility, and can be performed with little sample manipulation or processing.
Accordingly, it is an object of the present invention to provide a qualitative or quantitative assay for the detection of food or waterborne parasites such as C. parvum oocysts.
It is another object of the present invention to provide a detection assay for parasites, such as C. parvum oocysts, in either source or finished water.
It is yet another object of the present invention to provide a detection assay for parasites, such as C. parvum oocysts, in turbid samples, particularly turbid water samples.
It is another object of the present invention to provide a detection assay for food or waterborne parasites, such as C. parvum oocysts, in biological samples such as biological fluid samples and stool, or fecal samples.
Another object of the present invention is to provide a quantitative detection assay enabling the correlation of parasite levels in the food or water source, such as C. parvum oocyst levels in drinking water, and the incidence of disease in normal, healthy individuals, or immunocompromised individuals.
Yet another object of the present invention is to provide a method for solubilizing molecular markers of parasites, such as C. parvum oocysts.
Another object of the present invention is to provide a kit for an optimized assay configuration for automated point-of-use analysis for detecting parasites, such as C. parvum, in water, biological fluids, or fecal samples.
Another object of the present invention is to provide a method for the immunological detection of parasites, such as C. parvum oocysts, that utilizes electrochemiluminescence technology.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.